Edema induction by the disintegrin-like/cysteine-rich domains from a Bothrops atrox hemorrhagin

Viperine and crotaline snake venoms contain one or more hemorrhagic metalloproteases called hemorrhagins. The most potent hemorrhagins belong to the P-III class and have, in addition to the protease domain, disintegrin-like and cysteine-rich domains. Although proteolytic degradation of vascular endothelium basement membrane has been established to be the main factor responsible for hemorrhage, several studies reveal other factors that actually do facilitate this process. Recent evidence has shown that the nonprotease domains of the P-III class hemorrhagins are able to inhibit the platelet aggregation by blocking essential procoagulant integrins on platelets. In this study we report the identification of a hemorrhagin from Bothrops atrox venom. This enzyme, a P-III class metalloprotease, undergoes an apparent spontaneous degradation, releasing a proteic fragment containing the disintegrin-like/cysteine-rich domains. This fragment shows the capability to induce an edematogenic process, suggesting the existence of a still unknown nonenzymatic mechanism of vascular permeability increase.     

A novel metalloprotease from Gloydius halys venom induces endothelial cell apoptosis through its protease and disintegrin-like domains

A novel hemorrhagic metalloprotease, halysase, isolated from the snake venom of Gloydius halys induces apoptosis in endothelial cells. The purified metalloprotease is a monomeric glycoprotein with an isoelectric point of 4.8. Analysis of the cDNA sequence encoding halysase revealed that the enzyme consists of multifunctional domains including a proprotein domain, a protease domain, a disintegrin-like domain and a cysteine-rich domain. The metalloprotease has a DECD sequence in the disintegrin-like domain instead of the typical RGD sequence. Halysase strongly inhibits proliferation of human umbilical vein endothelial cells in a dose-dependent manner as well as adhesion of the cells to extracellular matrix proteins. The enzyme specifically hydrolyzes not only extracellular matrix proteins such as fibronectin, vitronectin, and type IV collagen, but also integrins alpha1beta1 and alpha5beta1. The apoptosis of endothelial cells induced by halysase is closely associated with activation of caspase-3 and decreased level of Bcl-X(L)/Bax. Apohalysase, which lacks metalloprotease activity, is also able to induce the apoptosis. Several lines of experimental evidence suggest that the protease domain and the disintegrin-like domain of halysase cooperatively contribute to the induction of endothelial cell apoptosis.     

A novel disintegrin-like domain of a high molecular weight metalloprotease inhibits platelet aggregation

Disintegrin is one of the functionally distinct domains in high molecular weight metalloproteases from various snake venoms and generally has an Arg-Gly-Asp (RGD) sequence that is recognized by specific cell surface integrins. A cDNA encoding the disintegrin-like domain of a snake venom metalloprotease was cloned, expressed in Pichia pastoris, and molecular function of the recombinant protein was characterized. The cDNA sequence indicated that the disintegrin-like domain contains an Asp-Glu-Cys-Asp (DECD) sequence in place of the RGD motif. The expressed disintegrin-like protein was designated as halydin and it was able to inhibit human platelet aggregation in a dose-dependent manner. Unlike other typical RGD-disintegrins, the recombinant non-RGD disintegrin, halydin, inhibited platelet aggregation by suppressing platelet adhesion to collagen rather than by blocking fibrinogen binding to glycoprotein (GP) IIb-IIIa on the platelet surface. Experimental evidence suggests that halydin binds to integrin alpha2beta1 on the platelet surface.     

Inhibition of platelet aggregation by the recombinant cysteine-rich domain of the hemorrhagic snake venom metalloproteinase, atrolysin A

The P-III class of venom metalloproteinases has, in addition to the proteinase domain, a disintegrin-like domain and a cysteine-rich domain. Recent evidence has shown that the nonproteinase domains of the P-III class of hemorrhagic metalloproteinases function in the inhibition of platelet aggregation by blocking essential procoagulant integrins on platelets. A specific role for the highly conserved cysteine-rich domain has yet to be described. In this study, we expressed the cysteine-rich domain from the hemorrhagic metalloproteinase atrolysin A and demonstrated its ability to inhibit collagen-stimulated platelet aggregation. Additionally, the cysteine-rich domain was shown to interact with MG-63 cells to inhibit adhesion to collagen I. These data suggest a functional role for the cysteine-rich domain of the P-III toxins in the observed coagulopathy by targeting the toxin to platelets and inhibiting collagen-stimulated platelet aggregation. These characteristics may function to synergistically increase the hemorrhagic effect of the toxins.     

Identification of sites in the cysteine-rich domain of the class P-III snake venom metalloproteinases responsible for inhibition of platelet function

Atrolysin A and jararhagin are class P-III snake venom metalloproteinases (SVMPs) with three distinct domains: a metalloproteinase, a disintegrin-like and a cysteine-rich. The metalloproteinase and the disintegrin-like domains of atrolysin A and jararhagin contain peptide sequences that interact with alpha2beta1 integrin and inhibit the platelet responses to collagen. Recently, the recombinant cysteine-rich domain of atrolysin A was shown to have similar effects, but the sequence(s) responsible for this is unknown. In this report, we demonstrate two complete peptide sequences from the homologous cysteine-rich domains of atrolysin A and jararhagin that inhibit both platelet aggregation by collagen and adhesion of alpha2-expressing K562 cells to this protein. In addition, the peptide effects on platelets do not seem to involve an inhibition of GPVI. These results identify, for the first time, sites in the cysteine-rich domain of SVMPs that inhibit cell responses to collagen and reveal the complexity of the potential biological effects of these enzymes with multifunctional domains.     

Complete amino acid sequence of kaouthiagin, a novel cobra venom metalloproteinase with two disintegrin-like sequences

The primary structure of kaouthiagin, a metalloproteinase from the venom of the cobra snake Naja kaouthia which specifically cleaves human von Willebrand factor (VWF), was determined by amino acid sequencing. Kaouthiagin is composed of 401 amino acid residues and one Asn-linked sugar chain. The sequence is highly similar to those of high-molecular mass snake venom metalloproteinases from viperid and crotalid venoms comprised of metalloproteinase, disintegrin-like, and Cys-rich domains. The metalloproteinase domain had a zinc-binding motif (HEXXHXXGXXH), which is highly conserved in the metzincin family. Kaouthiagin had an HDCD sequence in the disintegrin-like domain and uniquely had an RGD sequence in the Cys-rich domain. Metalloproteinase-inactivated kaouthiagin had no effect on VWF-induced platelet aggregation but still had an inhibitory effect on the collagen-induced platelet aggregation with an IC(50) of 0.2 microM, suggesting the presence of disintegrin-like activity in kaouthiagin. To examine the effects of these HDCD and RGD sequences, we prepared synthetic peptides cyclized by an S-S linkage. Both the synthetic cyclized peptides from the disintegrin-like domain and from the Cys-rich domain) had an inhibitory effect on collagen-induced platelet aggregation with IC(50) values of approximately 90 and approximately 4.5 microM, respectively. The linear peptide (RAAKHDCDLPELC) and the cyclized peptide had little effect on collagen-induced platelet aggregation. These results suggest that kaouthiagin not only inhibits VWF-induced platelet aggregation by cleaving VWF but also disturbs the agonist-induced platelet aggregation by both the disintegrin-like domain and the RGD sequence in the Cys-rich domain. Furthermore, our results imply that the corresponding part of the Cys-rich domain in other snake venom metalloproteinases also has a synergistic disturbing effect on platelet aggregation, serving as a second disintegrin-like domain. This is the first report of an elapid venom metalloproteinase with two disintegrin-like sequences.     

Molecular cloning and functional characterization of a snake venom metalloprotease.

A cDNA clone, MT-d, encoding metalloprotease precursor was isolated from snake (Agkistrodon halys brevicaudus) venom gland cDNA library. MT-d-I protein containing both metalloprotease and disintegrin domains, and MT-d-II protein containing the metalloprotease domain only were expressed in Escherichia coli and refolded successfully into their functional forms. Each of the refolded enzyme species exhibited distinct substrate specificity. Proteolytic activity of the MT-d-1 was able to hydrolyse type I gelatin, type-III and V collagens in contrast with the catalytic function of MT-d-II. MT-d-I protein having metalloprotease activity was also able to inhibit platelet aggregation. Functionally active MT-d-I protein underwent autoproteolytic processing in vitro to produce metalloprotease and disintegrin; this processing was accompanied by significant changes in the substrate specificity of the enzyme activity. Experimental evidence strongly suggests that the disintegrin domain in the metalloprotease precursor modulates the catalytic function of the enzyme in hydrolysing extracellular matrix proteins.     

Activation of leukocyte rolling by the cysteine-rich domain and the hyper-variable region of HF3, a snake venom hemorrhagic metalloproteinase

The functionality of the disintegrin-like/cysteine-rich domains of snake venom metalloproteinases (SVMPs) has been shown to reside in the cysteine-rich region, which can interact with VWA-containing proteins. Recently, the hyper-variable region (HVR) of the cysteine-rich domain was suggested to constitute a potential protein-protein adhesive interface. Here we show that recombinant proteins of HF3, a hemorrhagic P-III SVMP, containing the cysteine-rich domain (disintegrin-like/cysteine-rich and cysteine-rich proteins) but not the disintegrin-like protein were able to significantly increase leukocyte rolling in the microcirculation. Peptides from the HVR also promoted leukocyte rolling and this activity was inhibited by anti-alpha_M/beta₂ antibodies. These results show, for the first time, that the cysteine-rich domain and its HVR play a role in triggering pro-inflammatory effects mediated by integrins.     

Characterization of ''basparin A,'' a prothrombin-activating metalloproteinase,from the venom of the snake Bothrops asper that inhibits platelet aggregation and induces defibrination and thrombosis

A prothrombin activator, named 'basparin A,' was isolated from the venom of the crotaline snake Bothrops asper, the species responsible for the majority of snakebite cases in Central America. It is an acidic (pI 5.4), 70kDa, single chain P-III metalloproteinase comprising, in addition to the metalloproteinase domain, disintegrin-like, and high-cysteine domains. Basparin A is a glycoprotein displaying immunological cross-reactivity with BaH1, a P-III hemorrhagic metalloproteinase isolated from the same venom. It activates prothrombin through the formation of meizothrombin, without requiring additional cofactors; it is, therefore, a class A snake venom prothrombin activator. In contrast with most venom metalloproteinases, it does not degrade components of the extracellular matrix. Apart from its clotting activity, basparin A inhibits collagen-dependent platelet aggregation in vitro, an effect that does not depend on proteolytic activity. Clotting activity on human plasma is not abrogated by the plasma proteinase inhibitors alpha(2) macroglobulin and murinoglobulin, whereas activity is completely inhibited by Costa Rican polyvalent (Crotalinae) anti-venom. Basparin A does not induce local tissue alterations, such as hemorrhage, myonecrosis, and edema, in mice. Moreover, it does not induce systemic hemorrhage, thrombocytopenia nor prolongation of the bleeding time following intravenous administration. At low doses, the only observed effect induced by basparin A, when injected intravenously or intramuscularly into mice, is defibrin(ogen)ation. At higher doses, intravenous administration resulted in sudden death due to numerous occluding thrombi in pulmonary vessels. Basparin A is likely to play an important role in the coagulopathy associated with B. asper envenoming.     

Leberagin-C,A disintegrin-like/cysteine-rich protein from Macrovipera lebetina transmediterranea venom,inhibits alphavbeta3 integrin-mediated cell adhesion

Leberagin-C, a new member of the disintegrin-like/cysteine-rich (D/C) family, was purified to homogeneity from the venom of Tunisian snake Macrovipera lebetina transmediterranea. It is a monomeric protein with a molecular mass of 25,787 Da. Its complete sequence of 205 amino acid residues was established by cDNA cloning. The leberagin-C shows many conserved sequences with other known D/C proteins, like the SECD binding sites and a pattern of 28 cysteines. It is the first purified protein from M. lebetina transmediterranea with only two disintegrin-like/cysteine-rich domains. Leberagin-C is able to inhibit platelet aggregation induced by thrombin and arachidonic acid with IC₅₀ of 40 and 50 nM respectively. It was also able to inhibit the adhesion of melanoma tumour cells on fibrinogen and fibronectin, by interfering with the function of alphavbeta3 and, to a lesser extent, with alphavbeta6 and alpha5beta1 integrins. To our knowledge, leberagin-C is the sole described D/C protein that does not specifically interact with the alpha2beta1 integrin. Structure–activity relationship study of leberagin-C suggested that there are some important amino acid differences with jararhagin, the most studied PIII metalloprotease from Bothrops jararaca, notably around the SECD motif in its disintegrin-like domain. Other regions implicated in leberagin-C specificities could not be excluded.     

cDNA cloning and characterization of vascular apoptosis-inducing protein 1

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification from crude venom of Crotalus atrox of two vascular apoptosis-inducing proteins (VAP1 and VAP2) that specifically induce apoptosis in vascular endothelial cells. We report here the cDNA cloning and characterization of VAP1. VAP1 cDNA encoded a protein with 610 amino acid residues. The amino acid sequence predicted from the cDNA indicated that VAP1 belongs to the metalloprotease/disintegrin family and that it is a multidomain polypeptide with a proprotein domain, a metalloprotease domain, a disintegrin-like domain, and a cysteine-rich domain. In the disintegrin-like domain, the sequence DECD replaces the RGD sequence that has frequently been found in such domains. We demonstrated that VAP1 has Zn(2+)-dependent metalloprotease activity and degrades fibrinogen. After incubation in the presence of either EDTA or EGTA, VAP1 was hardly able to degrade fibrinogen and to induce apoptosis in VEC. Our results indicated that VAP1 is a new type of snake venom metalloprotease/disintegrin and suggest that the metalloprotease activity of VAP1 might be involved in the induction of apoptosis by VAP1 in VEC.     

Collagen binding is a key factor for the hemorrhagic activity of snake venom metalloproteinases

Snake venom metalloproteinases (SVMPs) are multifunctional enzymes involved in several symptoms following snakebite, such as severe local hemorrhage. Multidomain P-III SVMPs are strongly hemorrhagic, whereas single domain P-I SVMPs are not. This indicates that disintegrin-like and cysteine-rich domains allocate motifs that enable catalytic degradation of ECM components leading to disruption of capillary vessels. Interestingly, some P-III SVMPs are completely devoid of hemorrhagic activity despite their highly conserved disintegrin-like and cysteine-rich domains. This observation was approached in the present study by comparing the effects of jararhagin, a hemorrhagic P-III SVMP, and berythractivase, a pro-coagulant and non-hemorrhagic P-III SVMP. Both toxins inhibited collagen-induced platelet aggregation, but only jararhagin was able to bind to collagen I with high affinity. The monoclonal antibody MAJar 3, that neutralizes the hemorrhagic effect of Bothrops venoms and jararhagin binding to collagen, did not react with berythractivase. The three-dimensional structures of jararhagin and berythractivase were compared to explain the differential binding to collagen and MAJar 3. Thereby, we pinpointed a motif within the Da disintegrin subdomain located opposite to the catalytic domain. Jararhagin binds to both collagen I and IV in a triple helix-dependent manner and inhibited in vitro fibrillogenesis. The jararhagin-collagen complex retained the catalytic activity of the toxin as observed by hydrolysis of fibrin. Thus, we suggest that binding of hemorrhagic SVMPs to collagens I and IV occurs through a motif located in the Da subdomain. This allows accumulation of toxin molecules at the site of injection, close to capillary vessels, where their catalytic activity leads to a local hemorrhage. Toxins devoid of this motif would be more available for vascular internalization leading to systemic pro-coagulant effects. This reveals a novel function of the disintegrin domain in hemorrhage formation.     

Cloning, expression, and characterization of a cDNA encoding snake venom metalloprotease

A cDNA clone, MT-c, encoding metalloprotease was isolated from snake (Agkistrodon halys brevicadus) venom gland cDNA library. Deduced amino acid sequence indicated that MT-c is composed of a signal sequence, amino-terminal propeptide, a central metalloprotease domain, and a Lys-Gly-Asp (KGD) disintegrin domain. The partial cDNA encoding metalloprotease and disintegrin domain was subcloned and expressed in E. coli. The expressed MT-c protein was purified and successfully refolded into functional form retaining the enzyme activity. Analyses of the purified recombinant protease activity revealed that the enzyme hydrolyzes extracellular matrix proteins including type I gelatin, type IV and type V collagen, while type I, II, III collagens and fibronectin were insensitive to the proteolytic digestion. The recombinant enzyme was also able to degrade fibrinogen by specifically cleaving A alpha chain of the protein.     

A novel prothrombin activator from the venom of Micropechis ikaheka: isolation and characterization

A novel prothrombin activator, Mikarin, has been isolated from Micropechis ikaheka venom. It is a single polypeptide chain metalloproteinase with the apparent molecular weight of 47kDa. Mikarin exhibits Ca(2+)-independent prothrombin activation, but no effects on other blood coagulation factors, such as factor X and fibrinogen. Mikarin is the first member of group I prothrombin activators from elapid venom. Like other high-molecular-weight snake venom proteinases, it has three structural domains, metalloproteinase and disintegrin-like and Cys-rich domains, and belongs to the P-III class of snake venom metalloproteinases. The N-terminal of Mikarin exhibits 76% sequence identity with Cobrin, a metalloproteinase identified from Naja naja venom, but very lower identities were found when compared with those from viperid and crotalid venom. In addition, the presence of disintegrin-like and Cys-rich domains in snake venom metalloproteinases with diverse biological activities suggests that these domains may be important for their function.     

Snake venom proteases affecting hemostasis and thrombosis

The structure and function of snake venom proteases are briefly reviewed by putting the focus on their effects on hemostasis and thrombosis and comparing with their mammalian counterparts. Up to date, more than 150 different proteases have been isolated and about one third of them structurally characterized. Those proteases are classified into serine proteases and metalloproteinases. A number of the serine proteases show fibrin(ogen)olytic (thrombin-like) activities, which are not susceptible to hirudin or heparin and perhaps to most endogenous serine protease inhibitors, and form abnormal fibrin clots. Some of them have kininogenase (kallikrein-like) activity releasing hypotensive bradykinin. A few venom serine proteases specifically activate coagulation factor V, protein C, plasminogen or platelets. The venom metalloproteinases, belonging to the metzincin family, generally show fibrin(ogen)olytic and extracellular matrix-degrading (hemorrhagic) activities. A few venom metalloproteinases show a unique substrate specificity toward coagulation factor X, platelet membrane receptors or von Willebrand factor. A number of the metalloproteinases have chimeric structures composed of several domains such as proteinase, disintegrin-like, Cys-rich and lectin-like domains. The disintegrin-like domain seems to facilitate the action of those metalloproteinases by interacting with platelet receptors. A more detailed analysis of snake venom proteases should find their usefulness for the medical and pharmacological applications in the field of thrombosis and hemostasis.     

Purification and characterization of AHPM,a novel non-hemorrhagic P-IIIc metalloproteinase with α-fibrinogenolytic and platelet aggregation-inhibition activities,from Agkistrodon halys pallas venom

A novel non-hemorrhagic metalloproteinase, AHPM, was purified from the venom of Agkistrodon halys pallas by a combination of ion-exchange and gel filtration chromatography. AHPM is a dimeric glycoprotein with multiple pIs around pH 7.9 and has a molecular mass of 110 kDa with two blocked N-terminuses. Partial sequence of AHPM obtained by LC-MS/MS analysis together with its dimeric nature reveals that it is a P-IIIc snake venom metalloproteinase composed of metalloproteinase, disintegrin-like and cysteine-rich domains. AHPM has a conserved DECD sequence in the disintegrin-like domain. AHPM hydrolyzes casein and fibrinogen and also dissolves fibrin clots and the proteolytic activity is abolished by EDTA, but not by PMSF, suggesting that it is a metalloproteinase. The protease hydrolyzes rapidly the Aα-chain of fibrinogen followed by the Bβ-chain and does not cleave the γ-chain. AHPM contains endogenous Zn²⁺ and Ca²⁺ ions at a molar ratio of 1:1.9 and 1:4.2, respectively, and Zn²⁺ ions are essential for its proteolytic activity. AHPM inhibits collagen-and ADP-induced platelet aggregation with half maximal inhibitory concentrations of 200 ± 8 nM and 280 ± 10 nM, respectively. EDTA markedly attenuates the inhibition of ADP-induced platelet aggregation by AHPM, indicating that the fibrinogenolytic activity of AHPM is involved in its inhibition of ADP-induced platelet aggregation. AHPM is devoid of hemorrhagic activity when injected (up to 30 μg) subcutaneously into mice. AHPM is so far identified as first non-hemorrhagic P-IIIc SVMP which has both fibrinolytic and platelet aggregation-inhibition activities. The bifunctional enzyme may have a potential clinical application as a thrombolytic agent.     

Purification, cDNA cloning and characterization of the vascular apoptosis-inducing protein, HV1, from Trimeresurus flavoviridis.

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification and cDNA cloning from Crotalus atrox of a vascular apoptosis-inducing protein (VAP1) that specifically induces apoptosis in vascular endothelial cells. We report here the purification and cDNA cloning of another vascular apoptosis-inducing protein, HV1, from crude venom of Trimeresurus flavoviridis. The protein, namely HV1, was purified as an inducer of apoptosis in cultured vascular endothelial cells. HV1 was a homodimeric protein with a molecular mass of 110 kDa. HV1 cDNA encoded a protein with 612 amino-acid residues. The amino-acid sequence predicted from the cDNA was highly homologous to VAP1. The amino-acid sequence of HV1 indicated that HV1 belongs to the metalloprotease/disintegrin family, and that it is a multidomain polypeptide with a proprotein domain, a metalloprotease domain, a disintegrin-like domain and a cysteine-rich domain. In the disintegrin-like domain, the sequence DECD, replaces the RGD sequence that has frequently been found in such domains. This replacement also occurs in VAP1. Our results indicate HV1 as the first identified homolog of VAP1.     

Effects of three novel metalloproteinases from the venom of the West African saw-scaled viper, Echis ocellatus on blood coagulation and platelets

Two metalloproteinases, a 24-kDa P-I EoVMP1 and a 56-kDa P-III EoVMP2, have recently been isolated from the venom of the West African saw-scaled viper Echis ocellatus. We now reveal a new 65-kDa haemorrhagic group P-III metalloproteinase which we have designated EoVMP3. The aim of this study was to determine whether these three snake venom metalloproteinases (SVMPs) affect platelets and blood coagulation. EoVMP1 had no effect on the aggregation of washed human platelets, whereas EoVMP2 inhibited collagen-induced platelet aggregation. In contrast, EoVMP3 did not inhibit the aggregation of platelets by collagen but instead activated platelets in the absence of any additional co-factors. All three SVMPs were capable of activating prothrombin to varying degrees and can therefore be described as procoagulants. EoVMP1, EoVMP2 and EoVMP3 share sequence identity with other members of the reprolysin family, but differ greatly in their effects on some of the components that control haemostasis.     

Structures of two elapid snake venom metalloproteases with distinct activities highlight the disulfide patterns in the D domain of ADAMalysin family proteins

The structures of snake venom metalloproteases (SVMPs) are proposed to be useful models to understand the structural and functional relationship of ADAM (a disintegrin and metalloprotease) which are membrane-anchored proteins involved in multiple human diseases. We have purified, sequenced and determined the structures of two new P-III SVMPs – atragin and kaouthiagin-like (K-like) from Naja atra. Atragin exhibits a known C-shaped topology, whereas K-like adopts an I-shaped conformation because of the distinct disulfide pattern in the disintegrin-like (D) domain. K-like exhibits an enzymatic specificity toward pro-TNFα with less inhibition of cell migration, but atragin shows the opposite effect. The specificity of the enzymatic activity is indicated to be dominated mainly by the local structures of SVMP in the metalloprotease (M) domain, whereas the hyper-variable region (HVR) in the cysteine-rich (C) domain is involved in a cell-migration activity. We demonstrate also a pH-dependent enzymatic activity of atragin that we correlate with the structural dynamics of a Zn²⁺-binding motif and the Met-turn based on the structures determined with a pH-jump method. The structural variations between the C- and I-shapes highlight the disulfide bond patterns in the D domain of the ADAM/adamalysin/reprolysins family proteins.     

Disintegrin-like/cysteine-rich domains of the reprolysin HF3: Site-directed mutagenesis reveals essential role of specific residues

Little is known about the biochemical properties of the non-catalytic domains of snake venom metalloproteinases (SVMPs). The ECD sequence of the disintegrin-like domain (D-domain) has been assigned as the disintegrin motif and, recently, the hyper-variable region (HVR) of the cysteine-rich domain (C-domain) was suggested to constitute a potential protein-protein adhesive interface. Here we show that the recombinant C-domain of HF3, a hemorrhagic SVMP from Bothrops jararaca, as well as three peptides resembling its HVR, inhibit collagen-induced platelet aggregation, which indicates a role for the C-domain and its HVR in targeting HF3 to platelets. Site-directed mutagenesis was used for the first time to identify charged residues essential for the functionality of the disintegrin-like/cysteine-rich domains (DC-domains). Residues of the disintegrin loop (E467 and D469), and of the HVR (K568, K569 and K575) of HF3 were individually mutated to Ala. Interestingly, only the mutant D469A was obtained in soluble form in Escherichia coli and this single mutation caused loss of two functional activities of the DC-domains: inhibition of platelet aggregation and increase of leukocyte rolling in the microcirculation. In summary we demonstrate that the C-domain and its HVR are critical for HF3 to affect platelets and leukocytes, however, the disintegrin loop may be important for the functionality of the D-domain in the context of the C-domain.